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Recombinant human stem cell factor stimulates differentiation of mast cells
from dispersed human fetal liver cells
AM Irani, G Nilsson, U Miettinen, SS Craig, LK Ashman, T Ishizaka, KM Zsebo and LB Schwartz
Department of Pediatrics, Medical College of Virginia, Richmond 23298.
We have previously shown the development in vitro of tryptase+ human mast
cells from fetal liver cells cocultured with murine 3T3 fibroblasts. In
this study, recombinant human stem cell factor (rhuSCF), the ligand for the
c-kit proto-oncogene product called Kit, stimulated the growth and
differentiation primarily of mast cells from dispersed fetal liver cells,
whereas recombinant human interleukin-3 (rhuIL-3) stimulated the
differentiation of basophils along with other cell types. Cultures of fetal
liver cells were initiated and maintained in the presence of rhuSCF or
rhuIL-3 for up to 6 weeks. Metachromatic cells in cytospins were identified
as mast cells primarily on the basis of tryptase expression, and as MCT or
MCTC by immunohistochemistry using monoclonal antibodies against tryptase
and chymase, whereas basophils were metachromatic, polymorphonuclear, and
lacked these proteases. Levels of tryptase and histamine were measured by
radioimmunoassay, tryptase and chymase activities by peptide hydrolysis,
and cell surface Kit by flow cytometry with the monoclonal antibody YB5.B8.
The predominant presence of mast cells occurred only in the cultures
supplemented with rhuSCF. The percentage and total number of mast cells
increased over time with increasing concentrations of rhuSCF and reached a
plateau at 55 ng/mL. At this concentration of rhuSCF, mast cells first
appeared by day 7; by day 42, 106% of the starting number of cells were
present and 85% of these were tryptase+, 31% being weakly chymase+. These
mast cells appeared immature by ultrastructural criteria; most cells were
mononuclear, but some had nuclei with deeply divided lobes. DNA synthesis
in tryptase+ mast cells at days 21 and 28 of culture with rhuSCF was
demonstrated by incorporation of bromodeoxyuridine. Calculated levels of
histamine (1.2 pg/mast cell) and tryptase (0.9 pg/mast cell) were similar
to those determined previously in coculture experiments with murine 3T3
fibroblasts. Chymase activity was undetectable in most cell extracts. On
day 0, 4% to 20% of fetal liver cells expressed cell surface Kit. In the
presence of rhuSCF, the percentages and total numbers of Kit+ cells and the
apparent concentration of Kit per cell increased along with the number of
tryptase+ cells. In the presence of rhuIL-3, toluidine blue+, tryptase-
cells first and maximally appeared at day 14 (11% +/- 2.5%). The percentage
of these toluidine blue+ cells then declined to about 6% by days 21 and 35,
while the total number of positive cells declined over 10-fold. Kit+ cells
in the presence of rhuIL-3 declined from 9% on day 3 to 2% on day
35.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 80,
Issue 12,
pp. 3009-3021,
12/15/1992
Copyright © 1992 by The American Society of Hematology
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